Chimeric VEGF Peptides

ABSTRACT

Compositions for and methods of treating patients with malignancies associated with overexpression of VEGF, particularly ovarian cancer, are provided herein. The compositions include but are not limited to certain VEGF epitopes, multivalent peptides comprising the epitopes, and chimeric peptides comprising one or more of the epitopes and a T cell epitope.

This application is a continuation of U.S. application Ser. No.11/052,721, filed Feb. 7, 2005, now U.S. Pat. No. 8,080,253, whichclaims priority to U.S. Provisional Application No. 60/542,041, filedFeb. 5, 2004, the entire disclosure of which is incorporated herein byreference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compositions for and methods oftreating patients with malignancies associated with overexpression ofVEGF, particularly ovarian cancer. The compositions of the presentinvention include certain VEGF epitopes, multivalent peptides comprisingsaid epitopes, and chimeric peptides comprising one or more of saidepitopes and a T cell epitope.

BACKGROUND

Ovarian cancer is the most lethal gynecologic malignancy, with almost14,000 women in the United States expected to die of the disease in 2003[Jemal]. Unfortunately, there is no effective means for detection ofearly ovarian cancer, and as such over 75% of cases are diagnosed whenthe disease has spread to the upper abdomen or lymph nodes. Despiteintensive cytotoxic chemotherapy following radical surgery to reduceovarian cancer volume, the median survival of women with advanced andlarge-volume ovarian cancer is under 40 months [McGuire].

Recent studies have demonstrated the critical role of angiogenesis intumor development and the formation of metastatic tumor deposits. Theinhibition of tumor angiogenesis has emerged as a promising newtherapeutic modality. A number of biologic activities have beenidentified as being involved in this complex process, however, vascularendothelial growth factor (VEGF) is now known to be one of the mostpotent and specific pro-angiogenic factors responsible for tumor-inducedangiogenesis [Leung], and is the most promising target for inhibition oftumor-induced angiogenesis. VEGF is overexpressed in a number of humansolid malignancies, including ovarian cancer [Boocock, Olson]. VEGFoverexpression has also been demonstrated in women with ovarian cancerand has been shown to be a poor prognostic factor [Hollingsworth, Paley,Tempfer]. Thus, VEGF is a rational target against which immunization mayhave a role in the treatment or prevention of ovarian cancer.

Various strategies have been used to inhibit the function of VEGF. Theseinclude targeting the VEGF receptor (VEGFR), using gene therapytechniques that deliver antisense oligonucleotides, use of solubleVEGFR, development of receptor tyrosine kinase (RTK) inhibitors, andmonoclonal antibodies (Mab) directed against VEGF [Kim]. The mostpromising approach appears to be a recombinant humanized version of amurine anti-human VEGF Mab (rhuMab VEGF, Bevacizumab). This Mab has beentested in patients with metastatic cancer [Gordon, Margolin]. There are,however, several disadvantages to the use of antibody therapy.Importantly, passive immunization strategies involve the transfer ofantibody to the patient, and immunity is short-lived as the antibodiesare cleared from the circulation. Likewise, Mabs are often immunogenicthemselves, thereby limiting their long-term use. Also, large antibodyvolumes are necessary for effective sustained immunization.

The use of vaccines to prevent or treat ovarian cancer is a highlyattractive approach because of the expected minimal side effects ofvaccine therapy. Many cancers express tumor-associated antigens (TAA)that serve as targets for cancer vaccines. Strategies for immunizationhave included whole cell vaccines, protein and DNA vaccines, as well aspeptide vaccines; each type of antitumor vaccine has its advantages andlimitations. Peptides are an attractive anticancer vaccine in that theyare safe (free of pathogens and oncogenic potential), stable, easilyconstructed, and are a cost-effective vaccine system [Dakappagari,Peoples, Kaumaya]. Importantly, peptide vaccines lead to sustainedimmune responses and memory, unlike that from passive immunization.Limitations of peptide vaccines include the fact that unmodifiedpeptides are rarely immunogenic; thus rational peptide design isimperative to the development of an effective antitumor vaccine.

SUMMARY OF THE INVENTION

The present invention provides new compounds and compositions forstimulating the immune system and for treating malignancies associatedwith overexpression of the VEGF protein. The compounds are immunogenicepitopes of the human VEGF protein and human EG-VEGF protein, andchimeric and multivalent peptides that comprise such epitopes.

The first group of compounds are referred to hereinafter collectively as“VEGF epitopes.” The VEGF epitopes comprise from about 15 to about 50amino acids, more preferably from 17 to 40 amino acids, most preferablyfrom 18 to 35 amino acids. In one aspect, the VEGF epitope shown inTable 1 below or an antigenic or functional equivalent thereof:

TABLE 1 Immunogen Residues Amino Acid Sequence Secondary Structure VEGF126-143 KCECRPKKDRARQENPCG Turn-Helix-Tum (of SEQ ID NO: 1) EG-VEGF 50-67 CTPLGREGEECHPGSHKV Turn-Helix-Tum (of SEQ ID NO: 2)

In another aspect the VEGF epitope comprises amino acid 4 through aminoacid 21 of human VEGF (as shown below), amino acid 24-38 of human VEGF,amino acid 127 through amino acid 144 of human VEGF, amino acid 102through amino acid 122 of VEGF, amino acid 162 through amino acid 175 ofhuman VEGF, or amino acid 76 through amino acid 96 of VEGF.

The human VEGF sequence is:

(SEQ ID NO: 1) 1MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLD 60IFQEYPDEIEYIFKPSCVPLMRCGGCSNDEGLECVPTEESNITMQIMRIKPHQGQHIGEM 120SFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTHSRCKARQLELN 180ERTCRCDKPRR 190

In another aspect, the present VEGF epitope comprises amino acid 5through amino acid 15 of human EG-VEGF protein (as shown below), aminoacid 24 through amino acid 34 of human EG-VEGF, amino acid 50 throughamino acid 75 of human EG-VEGF, or amino acid 86 through amino acid 102of human EG-VEGF.

The human EG-VEGF sequence is:

(SEQ ID NO: 2) 1 MRGATRVSIMLLLVTVSDCAVITGACERDVQCGAGTCCAISLWLRGLRMCTPLGREGEECHPGSHKVPFFRKRKHHTCPCLPNLLCSRFPDG RYRCSMDLKNINF 105

The present invention also provides chimeric peptides, referred tohereinafter as “chimeric VEGF peptides”, which comprise at least one ofthe present VEGF epitopes or an antigenic or functional equivalentthereof. Preferably the chimeric VEGF peptides are from about 35 toabout 150, more preferably from about 35 to about 70 amino acids inlength. The chimeric VEGF peptides comprise three units. The first unitcomprises at least one VEGF epitope or an antigenic or functionalequivalent thereof. The second unit is a helper T (Th) cell epitope,preferably a promiscuous Th cell epitope. As used herein a “promiscuousTh cell epitope” is one that promotes release of cytokines that assistin bypassing MHC restriction. The second unit is from about 14 to about22, more preferably about 15 to 21, most preferably 16 amino acids inlength. Preferably, the Th cell epitope has one of the following aminoacid sequences:

SEQ ID NO: 3 N-S-V-D-D-A-L-I-N-S-T-I-Y-S-Y-F-P-S-V,, referredto hereinafter as “TT”; SEQ ID NO: 4P-G-I-N-G-K-A-I-H-L-V-N-N-Q-S-S-E,, referred to hereinafter as “TT1”;SEQ ID NO: 5 Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L,, referred tohereinafter as “P2”; SEQ ID NO: 6F-N-N-F-T-V-S-F-W-L-R-V-P-K-V-S-A-S-H-L-E,,referred to hereinafter as “P30”; SEQ ID NO: 7L-S-E-I-K-G-V-I-V-H-R-L-E-G-V,, referred to hereinafter as “MVF”;SEQ ID NO: 8 F-F-L-L-T-R-I-L-T-I-P-Q-S-L-N,, referred tohereinafter as “HBV”; SEQ ID NO: 9T-C-G-V-G-V-R-V-R-S-R-V-N-A-A-N-K-K-P-E,, referredto hereinafter as “CSP”.

The third unit of the chimeric peptide joins the first and secondpeptide units. The third unit is an amino acid or, preferably, a peptideof from about 2 to about 15 amino acids, more preferably from about 2 toabout 10 amino acids, most preferably from about 2 to about 6 aminoacids in length. The most preferred linker comprises the amino acidsequence Gly-Pro-Ser-Leu, SEQ ID NO: 10.

The present invention also provides multivalent VEGF peptides, whichcomprise a plurality, i.e., at least two of the present VEGF epitopes orfunctional equivalents thereof and a Th cell epitope. The VEGF epitopesand Th cell epitope are connected to a core β sheet template.Preferably, the template comprises two strands of alternating leucineand lysine residues, which are connected by a linker. The linker is anamino acid or, preferably, a peptide of from about 2 to about 15 aminoacids, more preferably from about 2 to about 10 amino acids, mostpreferably from about 2 to about 6 amino acids in length. The mostpreferred linker comprises the amino acid sequence Gly-Pro-Ser-Leu, SEQID NO: 10.

The present invention also relates to an immunogenic compositioncontaining a mixture of VEGF epitopes, a chimeric VEGF peptide, or amultivalent VEGF peptide and a pharmacologically acceptable carrier. Inone aspect, the carrier is a biodegradable microsphere. Such immunogeniccompositions are useful for treating malignancies with whichoverexpression of the VEGF protein is associated.

The present invention also relates to polynucleotides which encode atleast one of the VEGF epitopes described above. Such polynucleotides areuseful for producing the epitope by recombinant techniques. The presentinvention also relates to isolated polynucleotides having a sequencewhich encodes a chimeric VEGF cell peptide of the present invention.Such polynucleotides are useful for preparing the chimeric VEGF cellpeptide. Such polynucleotides are also useful in an immunogeniccomposition (e.g., DNA vaccine) for treating or preventing malignanciesin which overexpression of the VEGF protein is associated. Preferably,such immunogenic compositions are administered intramuscularly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows high-titered sera recognizing the B-cell epitope (VEGF) andimmunogen (MVF-VEGF) following active immunization with VEGF peptides.ELISA of New Zealand white rabbit sera against VEGF, demonstratingtiters>1:500,000 at 4 weeks following the second booster vaccination(3y+4).

FIG. 2 shows that VEGF peptide antibodies recognize rhVEGF by ELISA.Comparison of rabbit pre-immune sera, prior to vaccination (bar 1) andsera from rabbits vaccinated with VEGF peptides (bars 2-4). Bar 2 and 3represent rabbit 1 at 3 and 4 weeks after the second booster vaccination(3y+3 and 3y+4, respectively). Bar 4 represents sera from rabbit 2 at asimilar bleed.

FIG. 3 shows a Western blot of rhVEGF blotted with (A) VEGF peptideantibody or (B) Ab-4, a VEGF monoclonal antibody, demonstratingrecognition of the appropriate recombinant protein dimer at 42 kDa.

FIG. 4 shows that VEGF peptide antibodies disrupt the normal VEGF-VEGFRinteraction by flow cytometry using HUVECs, presumably through depletionof VEGF. (A) Evaluation of the positive (PC), negative (NC) andinhibitor antibody (IC) controls of Fluorokine® assay, and (B) the samePC and NC as in (A), and employing either mouse or rabbit VEGF peptideantibodies, both demonstrating disruption of the normal VEGF-VEGFRinteraction. The rabbit antibody labeled as “combo” represents rabbitVEGF peptide antibodies following immunization with both the MVF-VEGFimmunogen as well as another immunogen not described in thisinvestigation.

FIG. 5 shows that VEGF peptide antibodies disrupt angiogenesis intoMatrigel™. C57BL/6 mice were subcutaneously injected with Matrigel™incubated with rhVEGF with (FIG. 5A) or without (FIG. 5B) VEGFantibodies. After 10 days, the plugs were removed, stained, and bloodvessel invasion was counted. Compared with PBS control, addition of VEGFpeptide antibodies significantly disrupts angiogenesis in vivo.Magnification 40×, stained with Hoechst 33342.

FIG. 6 shows that VEGF peptide antibodies disrupt angiogenesis intoMatrigel™. C57BL/6 mice were subcutaneously injected with Matrigel™incubated with rhVEGF, with or without VEGF peptide antibodies. After 10days, the plugs were removed, stained, and blood vessel invasion wascounted. Compared with PBS control, addition of VEGF peptide antibodiessignificantly disrupts angiogenesis in vivo. Each bar represents themean (±SEM) of a group of three mice.

FIG. 7 is a graph showing rabbit anti-peptide antibodies againstimmunogenic epitopes of (A) VEGF and (B) EG-VEGF, with and without theaddition of the measles virus fusion protein (MVF).

FIG. 8 shows disruption of (A) and shortening of (B) cyclic estrouscycles as well as (C) a decrease in the number of primordial follicleswith passive immunization with anti-VEGF antibodies in a murine model.

FIG. 9 shows that (A) Anti-VEGF and (B) anti-EG-VEGF peptide antibodiesrecognize rhVEGF and rhEG-VEGF.

FIG. 10 shows Western blots of rhVEGF (A and B) blotted with (A)anti-VEGF peptide antibody and (B) Ab-4, a monoclonal anti-VEGFantibody. Western blot of rhEG-VEGF (C and D), blotted with (C) rabbitanti-EG-VEGF peptide antibody and (D) rabbit anti-VEGF/anti-peptideEG-VEGF “combination” antibody, all demonstrating recognition of theappropriate recombinant protein.

FIG. 11 shows the results of a Fluorokine assay for evaluation of thefunctional properties of anti-VEGF peptide antibodies. (A) Evaluation ofthe positive (PC), negative (NC) and inhibitor antibody (IC) controls ofFluorokine assay, and (B) the same PC and NC as in (A), and employingeither mouse or rabbit anti-VEGF peptide antibodies, as well as thecombination anti-VEGF/anti-EG-VEGF peptide antibody, all demonstratingdisruption of the VEGF-VEGF receptor interaction.

FIG. 12 shows injection of (A) subcutaneous matrigel and (B)subcutaneous matrigel combined with rhVEGF, stained withCD31:phycoerythrin conjugate antibody demonstrates increasedangiogenesis in C57/BL6 mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described by reference to moredetailed embodiments, with occasional reference to the accompanyingdrawings. This invention may, however, be embodied in different formsand should not be construed as limited to the embodiments set forthherein. Rather, these embodiments are provided so that this disclosurewill be thorough and complete, and will fully convey the scope of theinvention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for describing particularembodiments only and is not intended to be limiting of the invention. Asused in the description of the invention and the appended claims, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise. Allpublications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the following specification andattached claims are approximations that may vary depending upon thedesired properties sought to be obtained by the present invention. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should be construed in light of the number of significantdigits and ordinary rounding approaches.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Every numerical range given throughoutthis specification will include every narrower numerical range thatfalls within such broader numerical range, as if such narrower numericalranges were all expressly written herein.

Throughout this disclosure, reference will be made to compoundsaccording to the invention. Reference to such compounds, in thespecification and claims, includes esters and salts of such compounds.Thus, even if not explicitly recited, such esters and salts arecontemplated, and encompassed, by reference to the compounds themselves.

Additionally, as used herein, “peptide,” “polypeptide,” and “protein,”can and will be used interchangeably. “Peptide/polypeptide/protein” maybe used to refer to any of the three, but recitations of any of thethree contemplate the other two. That is, there is no intended limit onthe size of the amino acid polymer (peptide, polypeptide, or protein),that can be expressed using the present invention. Additionally, therecitation of “protein” is intended to encompass enzymes, hormone,receptors, channels, intracellular signaling molecules, and proteinswith other functions. Multimeric proteins can also be made in accordancewith the present invention.

While the naturally occurring amino acids are discussed throughout thisdisclosure, non-naturally occurring amino acids, or modified aminoacids, are also contemplated and within the scope of the invention. Infact, as used herein, “amino acid” refers to natural amino acids,non-naturally occurring amino acids, and amino acid analogs, all intheir D and L stereoisomers. Natural amino acids include alanine (A),arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine(Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I),leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P),serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V),hydroxyproline (O and/or Hyp), isodityrosine (IDT), and di-isodityrosine(di-IDT). Hydroxyproline, isodityrosine, and di-isodityrosine are formedpost-translationally. Use of natural amino acids, in particular the 20genetically encoded amino acids, is preferred.

The present invention provides peptides that are immunogenic epitopes ofthe human VEGF protein and the human EG-VEGF protein, referred tohereinafter collectively as “VEGF epitopes”.

The VEGF epitopes and their antigenic equivalents are capable ofinvoking a humoral response, which results in the production ofantibodies that are immunoreactive with the recombinant human VEGFprotein and/or human EG-VEGF protein. The VEGF epitopes encompasspeptides having one of the sequences, referred to hereinafter as the“reference sequences”, as described above. The reference sequences wereselected and scored using computer-aided analysis using six correlatesof antigenicity: (a) the profiles of chain flexibility and mobility ofindividual sequences was calculated according to Karplus and Schultz,Naturwiss 72:212-213, 1985; (b) hydropathy profiles were generated overa seven residue span setting and were finally smoothed with a threeresidue span using the scale of Kyte and Doolittle, J. Mol. Biol.157:105-132, 1982; (c) hydrophilicity profiles were generated over a6-residue window using the program of Hopp and Woods, Proc. Natl. Acad.Sci. USA 78:3824-3828, 1981; (d) analysis of the exposure of an aminoacid residue to water using a 1.4 Å probe) was carried out by thesolvent exposure algorithm of Rose, Science 229:834-838, 1985; (e)protrusion indices that predicts portions of proteins that areaccessible and protrude into the solvent were calculated by the methodof Thornton, EMBO J. 5:409-413, 1986; (f) the probability that a fiveresidue sequence is antigenic was determined by the method of Welling,FEBS Lett 188:215-218, 1985. The basic premise is that the algorithmsused in the predictions will always locate regions that aresurface-exposed on the protein and therefore most likely to be involvedin antibody binding.

Sequences were given a score of 1 to 6 based on their respective indexvalues and were ranked: the highest ranking sequences had the highestindividual score for the analyses examined (6/6), and successivecandidates had the next highest score (5/6), etc. The best scoringepitopes were further ranked by correlation with their secondarystructural attributes, e.g., an amphiphilic α-helical sequence or aβ-turn loop region are preferred over a random coil fragment. Computerprograms by Chou and Fasman, Adv. Enzymol. Relat. Subj. Biochem. 47:45-148, 1978 were used to predict the secondary structure (α-helix,β-strand/sheet, (3-turn/loop, random coil) and helical amphiphilicmoment. Electrostatic ion pairs and helix dipole interaction in helicalsegment were also considered (e.g., hydrophobic/hydrophilic balance).Preferably, the hydrophilic/hydrophobic balance is from 2/2 to 4/1.

As described herein, the VEGF cell epitopes also encompass peptides thatare antigenic and functional equivalents of the peptides describedabove. Such functional equivalents have an altered sequence in which oneor more of the amino acids in the corresponding reference sequence issubstituted, or in which one or more amino acids are deleted from oradded to the reference sequence. For example, cysteine residues may bedeleted or replaced with other amino acids to prevent formation ofincorrect intramolecular disulfide bridges upon renaturation.

While it is possible to have nonconservative amino acid substitutions,it is preferred that, except for the substitutions that are made toreplace cysteine, the substitutions be conservative amino acidsubstitutions, in which the substituted amino acid has similarstructural or chemical properties with the corresponding amino acid inthe reference sequence. By way of example, conservative amino acidsubstitutions involve substitution of one aliphatic or hydrophobic aminoacids, e.g., alanine, valine, leucine and isoleucine, with another;substitution of one hydroxyl-containing amino acid, e.g., serine andthreonine, with another; substitution of one acidic residue, e.g.,glutamic acid or aspartic acid, with another; replacement of oneamide-containing residue, e.g., asparagine and glutamine, with another;replacement of one aromatic residue, e.g., phenylalanine and tyrosine,with another; replacement of one basic residue, e.g., lysine, arginineand histidine, with another; and replacement of one small amino acid,e.g., alanine, serine, threonine, methionine, and glycine, with another.

Preferably, the deletions and additions are located at the aminoterminus, the carboxy terminus, or both, of one of the sequences shownabove. As a result of the alterations, the VEGF functional epitopeequivalent has an amino acid sequence which is at least 70% identical,preferably at least 80% identical, more preferably at least 90%identical, most preferably, at least 95% identical to the correspondingreference sequences. Sequences which are at least 90% identical have nomore than 1 alteration, i.e., any combination of deletions, additions orsubstitutions, per 10 amino acids of the reference sequence. Percentidentity is determined by comparing the amino acid sequence of thevariant with the reference sequence using MEGALIGN project in the DNASTAR program.

For functional equivalents that are longer than a correspondingreference sequence, it is preferred that the functional equivalent havea sequence which is at least 90% identical to the reference sequence andthe sequences which flank the reference sequence in the wild-type VEGFor EG-VEGF protein. In addition to being an antigenic equivalent of thenaturally-occurring human VEGF epitope, the functional equivalent isalso capable of raising antibodies that disrupt bind of human VEGF orEG-VEGF to the VEGF receptor.

Preparation of Epitopes and Co-Linear Chimeric Peptides

The VEGF epitopes, chimeric VEGF peptides, and multivalent VEGFpeptides, preferably, are synthesized using commercially availablepeptide synthesizers. Preferably, the chemical methods described inKaumaya et al., “DE NOVO” ENGINEERING OF PEPTIDE IMMUNOGENIC ANDANTIGENIC DETERMINANTS AS POTENTIAL VACCINES, in Peptides, Design,Synthesis and Biological Activity (1994), pp 133-164, which isspecifically incorporated herein by reference, are used.

The VEGF epitopes and chimeric peptides may also be produced usingcell-free translation systems and RNA molecules derived from DNAconstructs that encode the epitope or peptide. Alternatively, the VEGFepitopes or chimeric peptides are made by transfecting host cells withexpression vectors that comprise a DNA sequence that encodes therespective epitope or chimeric peptide and then inducing expression ofthe polypeptide in the host cells. For recombinant production,recombinant constructs comprising one or more of the sequences whichencode the epitope, chimeric peptide, or a variant thereof areintroduced into host cells by conventional methods such as calciumphosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape lading, ballistic introduction orinfection.

The VEGF epitopes and chimeric peptides may be expressed in suitablehost cells, such as for example, mammalian cells, yeast, bacteria,insect cells or other cells under the control of appropriate promotersusing conventional techniques. Suitable hosts include, but are notlimited to, E. coli, P. pastoris, Cos cells and 293 HEK cells. Followingtransformation of the suitable host strain and growth of the host strainto an appropriate cell density, the cells are harvested bycentrifugation, disrupted by physical or chemical means, and theresulting crude extract retained for further purification of the epitopeor chimeric peptide.

Conventional procedures for isolating recombinant proteins fromtransformed host cells, such as isolation by initial extraction fromcell pellets or from cell culture medium, followed by salting-out, andone or more chromatography steps, including aqueous ion exchangechromatography, size exclusion chromatography steps, and highperformance liquid chromatography (HPLC), and affinity chromatographymay be used to isolate the recombinant polypeptide. To produceglycosylated epitopes and chimeric peptides, it is preferred thatrecombinant techniques be used. To produce glycosylated epitopes andchimeric peptides which contain the same, it is preferred that mammaliancells such as, Cos-7 and Hep-G2 cells be employed in the recombinanttechniques.

Naturally occurring variants of the VEGF epitopes above may also beisolated by, for example, by screening an appropriate cDNA or genomiclibrary with a DNA sequence encoding the polypeptide.

Identifying Functional Equivalents of the VEGF Peptide

Functional equivalents of the VEGF epitopes shown above may generally beidentified by modifying the sequence of the epitope and then assayingthe resulting polypeptide for the ability to stimulate an immuneresponse, e.g., production of antibodies. For example, such assays maygenerally be performed by preparing a chimeric peptide which comprisesthe modified polypeptide and a promiscuous Th cell epitope, injectingthe chimeric peptide into a test animal and assaying for antibodies.Such antibodies may be found in a variety of body fluids including seraand ascites. Briefly, a body fluid sample is isolated from awarm-blooded animal, such as a human, for whom it is desired todetermine whether antibodies specific for the human VEGF are present.The body fluid is incubated with the human VEGF or EG-VEGF protein underconditions and for a time sufficient to permit immunocomplexes to formbetween the polypeptide and antibodies specific for the protein and thenassayed, preferably using an ELISA technique. In such technique, thecolorimetric change is measured at 490 nm. Epitopes which induceproduction of antibodies that exhibit a titer equal to 10,000 or greaterfor human VEGF or EG-VEGF protein, are preferred. As used herein a titerof 10,000 refers to an absorbance value of 0.2 above background.

Functional equivalents of the VEGF epitope are further identified bydetermining whether the peptide can be used to raise antibodies thatdisrupt binding of human VEGF to the human VEGF receptor as described inExample 1 below.

Determining Optimum Dosages for Inhibiting Agiogenesis or TreatingCancer

In vivo methods can also be used to characterize functional equivalentsof the present epitopes or to determine optimum dosages of the VEGFchimeric and multivalent peptides. In one aspect, a xenograft model canbe used. In this model, subcutaneous tumors are established with anovarian cancer (SKOV3) cell line by injecting BALB/c athymic nude micein the right flank with 2×10⁶ tumor cells mixed with matrigel. Tumorsare allowed to reach 150-200 mm³ and then mice randomized to receiveeither intraperitoneal injection of anti-VEGF or anti-EG-VEGF antibodiesor nonspecific IgG antibodies (control) every 3 days. Tumor volumes aremeasured and calculated twice weekly. Tumor permeability is assessedusing the Miles assay, in which treated and control mice undergointracardiac injection with Evans blue solution. The animals are then besacrificed, photographed, and the amount of permeability comparedbetween treated and control mice. The matrigel plug is removed, andvessel density examined by CD31 immunohistochemistry.

Active immunization with the VEGF and EG-VEGF peptide vaccines is theninvestigated in vivo. Previous investigation has shown that theintroduction of SKOV3 ovarian cancer cells (10×10⁶ cells in 200 μL PBS)in the peritoneal cavity of female immunodeficient mice (BALB/c nu/nu)leads to a clinical condition identical to that seen with women withadvanced ovarian cancer, including the establishment of tumors coatingthe surface of intraperitoneal organs, as well as the development oflarge-volume ascites within 4 weeks of inoculation [9]. Likewise,subcutaneous injection of SKOV3 cells (10×10⁶ cells in 50 μL PBS) leadsto measurable tumor within 10 days of inoculation. As such, acombination of both subcutaneous and intraperitoneal injection of SKOV3cells can be investigated in determining the in vivo efficacy of peptideimmunization against ovarian tumors.

Groups of 10 mice are vaccinated with the VEGF and EG-VEGF epitopesthree times at 3-week intervals. Antibody titers are monitored by ELISA.Three weeks following the third vaccination, mice are challenged withSKOV3 cells. Subcutaneous tumors are measured weekly using calipers, andtumor volumes are estimated based on the assumption that tumors arespherical. Because intraperitoneal tumor growth and distribution cannotbe measured directly, mice are killed by anesthetic overdoseapproximately 4 weeks following inoculation and location of tumor,presence of ascites, and tumor location and size recorded.

Further evidence of the anti-angiogenic effect of the elicited anti-VEGFantibodies is suggested by inhibition of ovulation by antibody followingstimulation by gonadotropins. Because ovulation is regulated throughangiogenic stimuli [18], it is believed that anti-angiogenic efficacy ofthe anti-peptide antibodies would lead to disruption of ovulation. Inthis experiment, groups of 24 day old female Sprague-Dawley rats areimmunized with anti-peptide antibodies against VEGF, EG-VEGF, both VEGFand EG-VEGF, or nonspecific antibody (control). Four hours followingimmunization, the rats are treated with 20 IU pregnant mares' serumgonadotropin (PMSG) and 24-hours later with 20 IU human chorionicgonadotropin (hCG), the hormonal stimulatory sequence that leads toovulation. After 5 days, the rats are killed and an assessment of theefficacy of inhibition of ovulation will be made. It is believed that inrats treated with anti-peptide antibodies, ovulation will be efficientlyinhibited and a decreased ovarian weight and alterations in the estrouscycle and decreased follicular growth and decreased CD31 immunostainingwill be seen compared with control animals.

Further assessment of the in vivo activity of VEGF peptide vaccines canbe conducted in a transgenic mouse model that expresses human VEGF underthe control of the rat insulin promoter (Rip1 VEGF-A) [20]. In thismodel, overexpression of VEGF was shown to accelerate the onset of tumorangiogenesis, and spontaneous tumors were shown to develop within 10weeks (single-transgenic mouse, RIP1Tag2) and 14 weeks(double-transgenic mouse, Rip1Tag2/Rip1 VEGF-A). In this studies, miceare immunized with VEGF or EG-VEGF epitopes on a compressed schedule tomatch the transgenic mouse tumor development (initial immunization willbe at 5 weeks, with subsequent booster at 7 and 9 weeks). Mice are thensacrificed, and tumor volumes calculated. Tumors are fixed, embedded inOCT, snap frozen, and analyzed by H&E staining. Vessel density candetermined by CD31 immunostaining. It is expected that VEGF peptidevaccination will lead to the prevention or inhibition of tumor formationin this transgenic mouse model overexpressing VEGF, and that surrogatesfor angiogenesis will be decreased.

Polynucleotides

The present invention also provides isolated polynucleotides whichencode the VEGF epitopes and the chimeric peptides of the presentinvention. The present polynucleotides also encompass polynucleotideshaving sequences that are capable of hybridizing to the nucleotidesequences of under stringent conditions, preferably highly stringentconditions. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, asdescribed in Berger and Kimmel (1987) Guide to Molecular CloningTechniques, Methods in Enzymology, vol 152, Academic Press. The term“stringent conditions,” as used herein, is the “stringency” which occurswithin a range from about Tm-5 (5° below the melting temperature of theprobe) to about 20° C. below Tm. As used herein “highly stringent”conditions employ at least 0.2×SSC buffer and at least 65° C. Asrecognized in the art, stringency conditions can be attained by varyinga number of factors such as the length and nature, i.e., DNA or RNA, ofthe probe; the length and nature of the target sequence, theconcentration of the salts and other components, such as formamide,dextran sulfate, and polyethylene glycol, of the hybridization solution.All of these factors may be varied to generate conditions of stringencywhich are equivalent to the conditions listed above.

Polynucleotides comprising sequences encoding a VEGF epitope or achimeric peptide of the present invention may be synthesized in whole orin part using chemical methods or, preferably, recombinant methods whichare known in the art. Polynucleotides which encode a VEGF may beobtained by screening a genomic library or eDNA library with antibodiesimmunospecific for them to identify clones containing suchpolynucleotide.

The polynucleotides are useful for producing a VEGF B epitope, chimericpeptide, or multivalent peptide. For example, an RNA molecule encoding achimeric peptide is used in a cell-free translation systems to preparesuch polypeptide. Alternatively, a DNA molecule encoding a VEGF epitopeor a chimeric peptide is introduced into an expression vector and usedto transform cells. Suitable expression vectors include for examplechromosomal, nonchromosomal and synthetic DNA sequences, e.g.,derivatives of SV40, bacterial plasmids, phage DNAs; yeast plasmids,vectors derived from combinations of plasmids and phage DNAs, viral DNAsuch as vaccinia, adenovirus, fowl pox virus, pseudorabies, baculovirus,and retrovirus. The DNA sequence is introduced into the expressionvector by conventional procedures.

Accordingly, the present invention also relates to recombinantconstructs comprising one or more of the present polynucleotidesequences. Suitable constructs include, for example, vectors, such as aplasmid, phagemid, or viral vector, into which a sequence that encodesVEGF epitope or the chimeric peptide has been inserted. In theexpression vector, the DNA sequence which encodes the epitope orchimeric peptide is operatively linked to an expression controlsequence, i.e., a promoter, which directs mRNA synthesis. Representativeexamples of such promoters, include the LTR or SV40 promoter, the E.coli lac or trp, the phage lambda PL promoter and other promoters knownto control expression of genes in prokaryotic or eukaryotic cells or inviruses. The expression vector, preferably, also contains a ribosomebinding site for translation initiation and a transcription terminator.Preferably, the recombinant expression vectors also include an origin ofreplication and a selectable marker, such as for example, the ampicillinresistance gene of E. coli to permit selection of transformed cells,i.e., cells that are expressing the heterologous DNA sequences. Thepolynucleotide sequence encoding the VEGF epitope or the chimericpeptide is incorporated into the vector in frame with translationinitiation and termination sequences. Preferably, the polynucleotidefurther encodes a signal sequence which is operatively linked to theamino terminus of the VEGF epitope, or chimeric peptide.

The polynucleotides encoding the VEGFF or EG-VEGF epitope or thechimeric peptides comprising such epitopes are used to expressrecombinant peptide using techniques well known in the art. Suchtechniques are described in Sambrook, J. et al (1989) Molecular CloningA Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. andAusubel, F. M. et al. (1989) Current Protocols in Molecular Biology,John Wile & Sons, New York, N.Y. Polynucleotides encoding the VEGF orEG-VEGF epitope or the chimeric peptides comprising such epitopes arealso used to immunize animals.

Pharmaceutical Compositions

Pharmaceutical compositions which comprise mixtures of VEGF and/orEG-VEGF epitopes, chimeric VEGF or EG-VEGF peptides, and multivalentVEGF- or EG-VEGF peptides or the polynucleotides which encode the sameare preferably formulated for use as a pharmaceutical composition (e.g.,an immunogenic composition or a vaccine). Such compositions generallycomprise one or more of the present VEGF and/or EG-VEGF epitopes, one ormore of the present VEGF and/or EG-VEGF chimeric peptides, or one ormore the present VEGF or EG-VEGF multivalent peptides or thepolynucleotides which encode the same in combination with apharmaceutically acceptable carrier, excipient, or diluent. Suchcarriers will be nontoxic to recipients at the dosages andconcentrations employed.

In addition to the epitopes, multivalent peptides, and chimeric peptides(which functions as antigens) or the polynucleotide which encodes thesame, other components, such as a vehicle for antigen delivery andimmunostimulatory substances designed to enhance the protein'simmunogenicity, are, preferably, included in the pharmaceuticalcomposition. Examples of vehicles for antigen delivery include aluminumsalts, water-in-oil emulsions, biodegradable oil vehicles, oil-in-wateremulsions, biodegradable microcapsules, and liposomes. For the vaccinesthat comprise the chimeric peptide, one potential vehicle for antigendelivery is a biodegradable microsphere, which preferably is comprisedof poly(D,L-lactide-co-glycolide) (PLGA).

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administrationand whether a substantial release is desired. For parenteraladministration, such as subcutaneous injection, the carrier preferablycomprises water, saline, alcohol, a fat, a wax, or a buffer.Biodegradable microspheres (e.g., polylactic galactide) may also beemployed as carriers for the pharmaceutical compositions of thisinvention. Optionally, the pharmaceutical composition comprises anadjuvant.

The VEGF epitope mixtures, chimeric and multivalent peptides and thepolynucleotides which encode the same are useful for enhancing oreliciting, in a subject or a cell line, a humoral response and,preferably, a cellular immune response (e.g., the generation ofantigen-specific cytolytic T cells). As used herein, the term “subject”refers to any warm-blooded animal, preferably a human. A subject may beafflicted with cancer, such as ovarian cancer, or may be normal (i.e.,free of detectable disease and infection). The pharmaceuticalcomposition is particularly useful for treating women who have a familyhistory of ovarian cancer or who have been diagnosed as having ovariancancer.

Methods of Treatment

The present invention also provides methods of treating a cancer whichis associated with overexpression of VEGF. By “treating” is meantinhibiting or slowing or retarding the growth of the tumor. Such cancersinclude ovarian cancer. The method comprises administering apharmaceutical composition comprising a VEGF and/or EG-VEGF epitopemixture, one or more VEGF chimeric peptides or one or more VEGFmultivalent peptides of the present invention to a subject. Preferablymultiple intramuscular injections, at three week intervals are used toadminister the pharmaceutical composition.

The present invention also provides methods of inhibiting angiogenesisin rapidly growing tissues in a subject. The methods compriseadministering a mixture of VEGF and/or EG-VEGF epitopes of the presentinvention, one or more chimeric VEGF and/or EG-VEGF chimeric peptides ofthe present invention, one or more multivalent VEGF and/or EG-VEGFpolypeptides of the present invention or polynucleotides that encode thesame to the subject.

The peptides of this invention relate to the representative peptides asdescribed above, and to antigenically related variants of thesepeptides. “Antigenically related variants” can be either naturalvariants or artificially modified variants that immunologically mimicthe VEGF or EG-VEGF epitope described above. Such artificially modifiedvariants can be made by synthetic chemistry of recombinant DNAmutagenesis techniques that are well known to persons skilled in the art(see for example Chapter 15 of Sambrook, et al. “Molecular Cloning aLaboratory Manual” (1989) Cold Spring Harbor Laboratory Press). Theantigenically related variants of the peptides should have an amino acidsequence identity of at least 75% to one of the VEGF or EG-VEGF epitopesdescribed above (and more preferably at least 85%, and most preferablyat least 95% identity), whilst still being capable of immunologicallymimicking the corresponding antigenic determinant site of the human VEGFor EG-VEGF protein.

For this invention “immunologically mimicking the correspondingantigenic determinant site of the VEGF or EG-VEGF protein is defined asa (variant) peptide being capable of inducing antibodies thatspecifically recognize one of the wild-type epitope sequences describedabove in the context of the whole VEGF or EG-VEGF protein AND/OR definedas a (variant) peptide being capable of being recognized by the sameimmunospecific antibody that recognizes one of the VEGF or EG-VEGFepitopes described above in the context of the whole VEGF or EG-VEGFprotein. In the first definition, the variant peptide should be capableof inducing such antibodies either by itself, or in conjunction with acarrier molecule. In the second definition, the variant peptide shouldbe capable of being recognized either by itself, or in conjunction witha carrier molecule. Antigenically related variants may have had aminoacids added, inserted, substituted or deleted. Preferred variants arethose that differ from the referents by conservative (preferably single)amino acid substitutions.

Polypeptides of the present invention can be prepared in any suitablemanner. Such polypeptides include recombinantly produced polypeptides,synthetically produced polypeptides, or polypeptides produced by acombination of these methods. Means for preparing such polypeptides arewell understood in the art, however examples of the method are presentedin the Examples section.

Polynucleotides of the Invention

The polynucleotides of the invention also relates to DNA sequences thatcan be derived from the amino acid sequences of the peptides andpolypeptides of the invention bearing in mind the degeneracy of codonusage. This is well known in the art, as is knowledge of codon usage indifferent expression hosts which is helpful in optimizing therecombinant expression of the peptides and polypeptides of theinvention.

The invention also provides polynucleotides which are complementary toall the above described polynucleotides.

When the polynucleotides of the invention are used for the recombinantproduction of polypeptides of the present invention, the polynucleotidemay include the coding sequence for the polypeptide, by itself; or thecoding sequence for the polypeptide in reading frame with other codingsequences, such as those encoding a leader or secretory sequence, apre-, or pro- or prepro-protein sequence, or other fusion peptideportions. For example, a marker sequence which facilitates purificationof the fused polypeptide can be encoded. In certain preferredembodiments of this aspect of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc Natl Acad Sci USA (1989) 86:821-824, oris an HA tag, or is glutathione-s-transferase. The polynucleotide mayalso contain non-coding 5′ and 3′ sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

Vectors, Host Cells, Expression

The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, and hostcells which are genetically engineered with vectors of the invention andto the production of peptides or polypeptides of the invention byrecombinant techniques. Cell-free translation systems can also beemployed to produce such proteins using RNAs derived from the DNAconstructs of the present invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Introduction of polynucleotides into hostcells can be effected by methods described in many standard laboratorymanuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986)and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)such as calcium phosphate transfection, DEAE-dextran mediatedtransfection, transfection, microinjection, cationic lipid-mediatedtransfection, electroporation, transduction, scrape loading, ballisticintroduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as meningococci, streptococci, staphylococci, E. coli, Streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used. Such systems include,among others, chromosomal, episomal and virus-derived systems, e.g.,vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression systems may contain control regions that regulate as well asengender expression. Generally, any system or vector suitable tomaintain, propagate or express polynucleotides to produce a polypeptidein a host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well known androutine techniques, such as, for example, those set forth in Sambrook etal., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of theendoplasmic, reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the desired polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Purification of Recombinantly Expressed Peptides/Polypeptides

Peptides and polypeptides of the invention can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulphate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxyapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding proteins may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Although the gene sequence of the chimeric VEGF polypeptide in thevector can be tagged with a Histidine-tag sequence which aids thepurification of the polypeptide, it is not an essential element to theinvention, as polypeptides without the Histidine-tag can still bepurified by one of the techniques mentioned above.

Antibodies

The peptides and polypeptides of the invention, or cells expressing themcan also be used as immunogens to produce antibodies immunospecific forthe wild-type VEGF or EG-VEGF. The term “immunospecific” means that theantibodies have substantially greater affinity for the peptides orpolypeptides of the invention than their affinity for other relatedpolypeptides in the prior art.

Antibodies generated against the peptides or polypeptides can beobtained by administering it to an animal, preferably a nonhuman, usingroutine protocols in the immunization of an animal with an antigen, thecollection of the blood, the isolation of the serum and the use of theantibodies that react with the peptide. The serum or IgG fractioncontaining the antibodies may be used in analyzing the protein. Forpreparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler, G. and Milstein, C.,Nature (1975) 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today (1983) 4:72) andthe EBV-hybridoma technique (Cole et al., MONOCLONAL ANTIBODIES ANDCANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can also be adapted to produce single chain antibodies topeptides or polypeptides of this invention. Also, transgenic mice, orother organisms including other mammals, may be used to expresshumanized antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the peptide or to purify the peptides or polypeptidesof the invention by affinity chromatography.

Vaccines

Another aspect of the invention is a vaccine composition comprising animmunogenic amount of at least one peptide or polypeptide of theinvention. Preferably the composition should also comprise apharmaceutically acceptable excipient. Vaccine preparation is generallydescribed in Vaccine Design (“The subunit and adjuvant approach” (eds.Powell M. F. & Newman M J). (1995) Plenum Press New York).

Additionally, the peptides and polypeptides of the present invention arepreferably adjuvanted in the vaccine formulation of the invention.Suitable adjuvants include an aluminum salt such as aluminum hydroxidegel (alum) or aluminum phosphate, but may also be a salt of calcium,iron or zinc, or may be an insoluble suspension of acylated tyrosine, oracylated sugars, cationically or anionically derivatizedpolysaccharides, or polyphosphazenes. Other known adjuvants include CpGcontaining oligonucleotides. The oligonucleotides are characterized inthat the CpG dinucleotide is unmethylated. Such oligonucleotides arewell known and are described in, for example W096/02555.

Further preferred adjuvants are those which induce an immune responsepreferentially of the TH1 type. High levels of Th1-type cytokines tendto favor the induction of cell mediated immune responses to the givenantigen, whilst high levels of Th2-type cytokines tend to favor theinduction of humoral immune responses to the antigen. Suitable adjuvantsystems include, for example monophosphoryl lipid A, preferably3-de-O-acylated monophosphoryl lipid A (3D-MPL), or a combination of3DMPL together with an aluminum salt. CpG oligonucleotides alsopreferentially induce a TH1 response. An enhanced system involves thecombination of a monophosphoryl lipid A and a saponin derivativeparticularly the combination of QS21 and 313-MPL as disclosed in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol as disclosed in WO 96/33739. A particularly potentadjuvant formulation involving QS21 3D-MPL & tocopherol in an oil inwater emulsion is described in WO 95/17210 and is a preferredformulation.

Another aspect of the invention relates to a method for inducing animmunological response in a mammal which comprises inoculating themammal with a peptide or polypeptide of the invention adequate toproduce antibody to inhibit angiogenesis and to inhibit growth of tumorsamong others. Yet another aspect of the invention relates to a method ofinducing immunological response in a mammal which comprises, deliveringa peptide or polypeptide of the invention via a vector directingexpression of a polynucleotide of the invention in vivo in order toinduce such an immunological response to produce antibody to protectsaid animal from diseases.

A further aspect of the invention relates to an immunological/vaccineformulation (composition) which, when introduced into a mammalian host,induces an immunological response in that mammal to VEGF or EG-VEGFpeptide wherein the composition comprises a polynucleotide encoding aVEGF or EG-VEGF epitope or the VEGF or EG-VEGF epitope itself. Thevaccine formulation may further comprise a suitable carrier. The VEGFvaccine composition is preferably administered orally, intranasally orparenterally (including subcutaneous, intramuscular, intravenous,intradermal, transdermal injection). Formulations suitable forparenteral administration include aqueous and non-aqueous sterileinjection solutions which may contain anti-oxidants, buffers,bacteriostats, and solutes which render the formulation isotonic withthe blood of the recipient; and aqueous and non-aqueous sterilesuspensions which may include suspending agents or thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example, sealed ampoules and vials and may be stored in afreeze-dried condition requiring only the addition of the sterile liquidcarrier immediately prior to use. The vaccine formulation may alsoinclude adjuvant as described above. The dosage will depend on thespecific activity of the vaccine and can be readily determined byroutine experimentation.

Yet another aspect relates to an immunological/vaccine formulation whichcomprises the polynucleotide of the invention. Such techniques are knownin the art, see for example Wolff et al., Science, (1990) 247: 1465-8.

EXAMPLES

Exemplary methods are described below, although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present peptides, compositions and methods.All publications and other references mentioned herein are incorporatedby reference in their entirety. The materials, methods, and examples areillustrative only and not intended to be limiting.

Example 1

Abstract

VEGF epitopes were identified using a computer-aided analysis employingspecific correlates for antigenicity. These epitopes were synthesized,purified, and combined the measles virus fusion (MVF) protein, a T-cellepitope. VEGF peptide antibodies were elicited in mice and rabbitsfollowing primary and booster vaccination. ELISA and Western blottingdetermined antibody specificity. VEGF peptide antibody function wasassessed by Fluorokine® VEGF receptor interaction assay. Vascularinvasion, a marker of angiogenesis, was determined by injecting C57BL/6mice subcutaneously with Matrigel™ (a mouse sarcoma-derived basementmembrane) incubated with and without recombinant human VEGF protein inthe presence or absence of VEGF peptide antibodies and mouse VEGFmonoclonal antibodies. Angiogenesis into the Matrigel™ assessed withHoechst staining, and angiogenesis was determined quantitatively bycounting blood vessels that invaded into the Matrigel™. Differences inrelative angiogenesis were compared with Student's t-test.

VEGF peptides from the predicted antigenic region elicited high-titerantibodies in mice and rabbits (1:500,000 at 3y+4). These antibodieswere demonstrated to be specific for rhVEGF by ELISA and Western blotanalysis. In vitro, the VEGF peptide antibodies led to a significantdisruption of the normal interaction between VEGF and the VEGF receptorcompared with control by the Fluorokine® assay. In vivo, the Matrigel™invasion assay revealed that VEGF peptide antibodies led to a profoundquantitative decrease in blood vessel invasion into the matrix comparedwith control (vascular invasion of VEGF alone=118.2 versus VEGF+VEGFpeptide antibodies=26.4, P=0.005).

Conclusion: VEGF peptide antibodies elicited from antigenic regions ofVEGF are immunogenic, specific, and anti-angiogenic. Thus, activeimmunization with a VEGF peptide vaccine is expected to be abiologically relevant treatment in women with epithelial ovarian cancer.

Materials and Methods

VEGF Epitope Selection

The selection of candidate VEGF 8-cell epitopes was performed usingcomputer-aided analysis using specific correlates for antigenicity(Peptide Companion, PeptiSearch), employing the profiles of chainflexibility and motility, hydropathy, protrusion indices, andantigenicity [Kaumaya 1994]. Sequences were given a score of 1 to 6based on their respective index values and were ranked. The best scoringepitopes were further ranked by correlation with their secondarystructural attributes, where an amphiphilic alpha-helical sequence or abeta-turn loop is preferred over random coil fragments. Finally,consideration was given to the individual amino acid sequence.Electrostatic ion pairs and helix dipole interaction in helical segmentwere also considered (hydrophobic/hydrophilic balance). The sequencesreceiving the highest scores were selected for further investigation.Our group has evaluated a number of antigenic peptides containing T-cellepitopes derived from non-human sources that have been identified to be“promiscuous” in their recognition in association with many MHCmolecules and their capacity to elicit T_(H) responses [Kaumaya 1993].Measles virus fusion (MVF) protein sequence 288-302 was chosen as thepromiscuous epitope to overcome the challenge of tolerance and MHCpolymorphism. The MVF epitope was linearly joined to the VEGF epitope bya four-residue linker (GPSL) on a peptide synthesizer. The glycine andproline residues in the linker potentiate a beta-turn in theoligopeptide, whereas the serine favors hydrogen bonds with the free FINof the backbone. The flexible nature of the linker allows forindependent folding of the T- and B-cell epitopes. Peptides werepurified by reverse-phase HPLC to ensure >95% purity. The identity ofthe peptides was performed by matrix-assisted LASER desorptionionization-time of flight spectrometry (MALDI-TOF).

Vaccination and Elicitation of VEGF Peptide Antibodies

To generate VEGF antibodies, New Zealand white rabbits (Charles RiverLaboratories, Inc., Wilmington, Mass.) and BALB/c mice (Harlan,Indianapolis, Ind.) were immunized subcutaneously at multiple sites witha total of 1 mg of each peptide emulsified in a Squaline/Arlacel vehiclecontaining nor-MDP (N-acetyl-glucosamine-3 yl-acetylL-alanyl-D-isoglutamine). Subsequent booster injections were given at 3weeks (secondary immunization, 2y) and 6 weeks (tertiary immunization,3y) after primary (1y) immunization. Rabbit and mouse sera werecollected weekly, and complement was inactivated by heating to 56° C.High-titered sera were purified on a protein A/G-agarose column (Pierce,Rockford, Ill.), and eluted antibodies were concentrated and exchangedin PBS using Mr 100,000 cutoff centrifuge filter units (Millipore,Bedford, Mass.). Antibody concentration was quantified by ELISA.

Characterization of VEGF Peptide Antibodies

Western blot analysis was undertaken to determine whether the VEGFpeptide antibodies recognize the VEGF protein. Proteins, includingrhVEGF, were resolved by 15% SDS-PAGE, transferred to nitrocellulose,and probed with VEGF peptide antibodies or a mouse VEGF monoclonalantibody (Ab-4, Neoprobe, Inc., Fremont, Calif.). Protein transfer wasmonitored with prestained molecular weight standards. Immunoreactivebands were detected by enhanced chemiluminescence (Pierce Biotechnology,Inc., Rockford, Ill.) using horseradish peroxidase-conjugated goatanti-rabbit immunoglobulins.

In an effort to determine the effect of our VEGF peptide antibodies onthe interaction of VEGF with the VEGF receptor, we employed theFluorokine® assay (R&D Systems, Minneapolis, Minn.). In brief, 5×10⁵HUVECs were washed and incubated with the biotinylated rhVEGF that inturn binds to the cells via the VEGFR. The cells are then directlyincubated with avidin-fluorescein, which attaches to the receptor-boundbiotinylated VEGF. Unbound biotinylated cytokine participates in anamplification reaction with the bound cytokine that results in anenhanced signal without compromising specificity. Cells expressing theVEGFR are fluorescently stained, with the intensity of stainingproportional to the density of the receptors. Relative receptor densityis then determined by flow cytometry. Through this experiment, weinitially standardized the flow cytometry component of the assay, inwhich different cell populations were identified using the suppliedpositive (rhVEGF) and negative (no stimulation) controls, as well aswith an inhibitory antibody provided with the kit. Followingstandardization, various concentrations of mouse or rabbit VEGF peptideantibodies were used instead of the supplied inhibitory antibody todetermine the proportional VEGFR density.

Determination of Anti-Angiogenic Properties of VEGF Peptide Antibodies

Analysis of blood vessel invasion was determined in a mouse modelemploying Matrigel™, a solubilized basement membrane matrix extractedfrom EHS mouse sarcoma [Passiniti]. Matrigel™ is a liquid at 4° C., butpolymerizes at 4° C., thus allowing for its removal from an animal hostfor analysis. Sets of 5 female C57BL/6 mice (Harlan, Indianapolis, Ind.)were injected subcutaneously with a total volume of 500 μL includingMatrigel™, various concentrations of rhVEGF, and various concentrationsof antibody (either VEGF monoclonal antibody (MAB293, R&D Systems,Minneapolis, Minn.) or VEGF peptide antibodies). After 10 days, the micewere sacrificed. The Matrigel™ plugs were then removed, sectioned, andstained with hematoxylin and eosin and the nuclear stain Hoechst 33342.Invasion into the plug was determined using an inverted fluorescentmicroscope at 40× magnification. Blood vessels at the periphery of theplug were identified and counted in a circumferential manner around theplug, and counted using computer-aided analysis in a blinded fashion.Statistical comparisons between groups were made using the Student'st-test.

Results

Computer-aided analysis of candidate B-cell epitopes of VEGF was used toselect residues 126-143 of SEQ ID NO: 1 (KCECRPKKDRARQENPCG), whichcorrelates with a secondary structure of turn-helix-turn, as beingpotentially immunogenic and antigenic. This epitope was linearly joinedto the promiscuous T-cell epitope of the measles virus protein (MVF)with the four-residue GPSL linker on a peptide synthesizer. Peptideswere purified, and their identity confirmed by MALDI-TOF. Rabbits andmice were immunized subcutaneously with the MVF-VEGF immunogen, and serawas obtained and purified. High VEGF peptide antibody titers (1:500,000at 4 weeks following tertiary immunization, 3y+4) were identified byELISA as demonstrated in rabbits in FIG. 1.

ELISA (FIG. 2) and Western blot (FIG. 3A) demonstrate that VEGF peptideantibodies recognize the rhVEGF protein. Ab-4, a monoclonal antibodyagainst VEGF, was used as a positive control in the Western blot, andconfirmed the expected rhVEGF protein homodimer at 42 kDa (FIG. 3B).

Following demonstration of the antigenic and immunogenic properties ofthe VEGF peptide antibodies, the functional properties of the antibodieswere evaluated. These VEGF peptide antibodies were demonstrated tosignificantly disrupt the normal interaction between VEGF and the VEGFreceptor (VEGFR) as determined by the Fluorokine® assay (FIG. 4). Inthis experiment, the addition of the VEGF peptide antibodies leads tobinding of VEGF, thus leading to a decrease in the normal VEGF-VEGFRinteraction.

We went on to determine whether these VEGF peptide antibodies hadanti-angiogenic properties as a result of inhibition of VEGF function.When compared with subcutaneous plugs of Matrigel™ incubated with rhVEGFprior to injection in C57BL/6 mice, plugs concurrently incubated withrhVEGF and VEGF peptide antibodies demonstrated significantly decreasedangiogenesis into the Matrigel™ (P=0.005, FIGS. 5 and 6). Theanti-angiogenic properties of the VEGF peptide antibodies wereequivalent to that of the VEGF monoclonal antibody used as a positivecontrol (P=NS comparing VEGF peptide antibodies to VEGF monoclonalantibodies, data not shown).

Discussion

We demonstrate that with rational peptide design employing VEGF B-cellepitopes, VEGF-specific autoantibodies are elicited. These antibodiesrecognized the full length protein from which the peptide was designed,and inhibit the expected protein function. Immunotherapy for cancertreatment has evolved substantially over the past decades. Previously,patients were treated with nonspecific immune stimulants, whereascurrently therapy is focused on identifying specific tumor-associatedantigens (TAAs) as targets for immunotherapy. Tumor-specificimmunotherapy can be categorized into passive, where antibodies aretargeted directly to tumor cells, and active, where vaccination withpeptides, tumor cells, tumor cell lysates, carbohydrates, geneconstructs, and anti-idiotype antibodies that mimic TAAs are employed ina host that mounts a specific immune response.

Historically, active immunization with peptides has had limited efficacybecause of their limited immunogenicity. Antibodies elicited in animalsby immunization with synthetic peptides have generally been shown tohave low affinity for the native protein, partly because antibodyrecognition sites are usually conformational, and the peptide immunogenslacked defined structure in solution. The genetically restrictedstimulatory activity of peptides was also a major obstacle to developingvaccine approaches for use in an outbred human population [Dulofeut].Covalent conjugation of B-cell epitope peptides to large carriermolecules was sometimes used to address this problem but often resultedin hypersensitivity, conformational changes, appearance of undefinedstructures, loss of epitopes, inappropriate presentation of epitopes,and batch-to-batch conjugate variability. We have addressed several ofthese issues in our rational approach to subunit peptide vaccine design[Dakappagari].

Our strategy involved de novo design of topographic determinants thatfocused on preserving the native protein sequence while facilitatingfolding of the peptide into a stable conformation that mimics the nativeprotein structure [Kobs, Kaumaya 1990]. We have demonstrated theeffectiveness of incorporating promiscuous T-helper epitopes derivedfrom nonhuman molecules into these constructs to overcome human MHCgenetic polymorphism [Kaumaya 1993]. Our previous work in a variety ofmodel systems has demonstrated that this approach can elicit high-titerantibodies that recognize native protein in an outbred population, andis confirmed in this investigation of VEGF epitopes.

Importantly, subunit peptide vaccines can focus immune responses tobiologically active epitopes. The need for epitope-based vaccines stemsfrom the fact that tolerance to self-antigens, such as VEGF, may limit afunctional immune response to whole protein-based vaccines due toactivation of suppressor T cells that maintain tolerance to hostantigens or alternate regulatory mechanisms [Sakaguchi]. The capacity tonarrowly focus the immune response is of particular relevance to VEGF,where interaction of the antibody with specific sites has the potentialof inhibiting growth. In contrast to passive therapy, the continuousavailability of tumor-targeting antibodies can be ensured at low cost.

Previous investigators have developed similar strategies of anti-VEGFcancer therapy. Interest in VEGF as a model antigen to exploreimmunogene therapy has been demonstrated through the construction of aplasmid DNA encoding Xenopus homologous VEGF [Wei]. This groupdetermined that immunogene tumor therapy with this vaccine led to thedevelopment of VEGF-specific antibodies that were anti-angiogenic andinhibited tumor formation. Importantly, treatment of mice with theimmunogene led to no significant toxic effects. In other work,vaccination with dendritic cells transfected with VEGF mRNA has beendemonstrated to lead to cytotoxic T lymphocyte (CTL) responses, to thedisruption of angiogenesis, and to antitumor efficacy withoutsignificant morbidity or mortality in vivo in a murine model [Nair].Thus, previous work has demonstrated the feasibility of activeimmunization using VEGF as a TAA.

Limitations of this investigation are the fact that the antigen chosenfor investigation, VEGF, is ubiquitously expressed in normal andpathologic conditions, and its inhibition may lead to potentiallyserious biologic consequences. Although fetal development is stronglycontrolled by angiogenesis, only reproduction, wound healing and cancerare controlled by angiogenesis in the adult host. As such, we believethat the relative control and expression of VEGF overexpression inmalignancy would lead to an acceptable therapeutic ratio in thetreatment of solid tumors. This is supported by previous investigationof other methods of decreasing the effects of VEGF (i.e. through DNAvaccines or inhibition of the VEGFR) that failed to demonstratesignificant toxicity.

Most women with ovarian cancer are diagnosed with advanced disease, anddespite the majority obtaining a complete clinical response followinginduction chemotherapy, 80% will recur and succumb to their disease.This scenario suggests that microscopic residual disease after initialtherapy is responsible for disease recurrence. For this reason, acurrent clinical research focus in the treatment of ovarian cancer isthe consideration of maintenance chemotherapy. Here, following initialtreatment, patients achieving a complete clinical response have beendemonstrated to have a better disease-free survival when a prolongedcourse of treatment is initiated immediately [Markman].

Interestingly, investigation of the role of active immunization with theanti-idiotype antibody ACA125 (which imitates the tumor-associatedantigen CA125 in ovarian cancer) as a maintenance chemotherapy inovarian cancer has demonstrated a positive impact on overall survival[Wagner]. Thus, active immunization as maintenance chemotherapy toprevent symptomatic recurrence of ovarian cancer is an attractiveconcept. Angiogenesis has been demonstrated to influence cancer growthvariably at different stages of malignant proliferation. Importantly,premalignant neoplastic conditions and small malignant tumors arethought to grow under the direct influence of endothelial mitogens suchas VEGF, whereas larger malignant tumors may grow and metastasizeindependent of angiogenic factors [Hanahan and Folkman]. The conceptthat angiogenic factors control early tumor growth has been applied tothe clinical management of ovarian cancer. Current research efforts aredirected at investigating chemotherapy agents that may act asanti-angiogenic, cytostatic agents. These compounds, such as tamoxifenand thalidomide, are being evaluated in women with early recurrent,asymptomatic ovarian cancer to determine if anti-angiogenic therapy mayprevent the development of clinically significant, symptomatic disease.As such, anti-angiogenic therapy with active immunization using VEGFepitopes could serve as a rational maintenance therapy that couldsignificantly impact the treatment of ovarian cancer.

From this investigation, we demonstrate that rational design of peptidevaccines against VEGF leads to elicitation of high-titered VEGF peptideantibodies that are specific and anti-angiogenic.

CITED DOCUMENTS FOR EXAMPLE 1

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Example 2

Selection of VEGF and EG-VEGF Epitopes

The selection of candidate VEGF and EG-VEGF B-cell epitopes has beenperformed using computer-aided analysis using specific correlates forantigenicity employing the profiles of chain flexibility and motility,hydropathy, protrusion indices, and antigenicity. Sequences were given ascore of 1 to 6 based on their respective index values and were ranked.The best scoring epitopes were further ranked by correlation with theirsecondary structural attributes, where an amphiphilic alpha-helicalsequence or a beta-turn loop is preferred over random coil fragments.Finally, consideration was given to the individual amino acid sequence.Electrostatic ion pairs and helix dipole interaction in helical segmentwere also considered (hydrophobic/hydrophilic balance). The sequencesreceiving the highest scores were selected for further investigation.Table 1 lists the sequences and secondary structure for VEGF and EG-VEGFepitopes selected for investigation.

Our group has evaluated a number of antigenic peptides containing T-cellepitopes derived from non-human sources that have been identified to be“promiscuous” in their recognition in association with many MHCmolecules and their capacity to elicit T_(H) responses. Measles virusfusion (MVF) protein sequence 288-302 was chosen as the promiscuousepitope to overcome the challenge of tolerance and MHC polymorphism. TheMVF epitope was linearly joined to the VEGF or EG-VEGF epitope by afour-residue linker (GPSL) (SEQ ID NO: 10) on a peptide synthesizer. Theglycine and proline residues in the linker potentiate a beta-turn in theoligopeptide, whereas the serine will favor hydrogen bonds with the freeFIN of the backbone. The flexible nature of the linker allows forindependent folding of the T- and B-cell epitopes. Peptides werepurified by reverse-phase HPLC to ensure >95% purity. The identity ofthe peptides was performed by matrix-assisted LASER desorptionionization-time of flight spectrometry.

To generate anti-VEGF and anti-EG-VEGF antibodies, rabbits wereimmunized subcutaneously at multiple sites with a total of 1 mg of eachpeptide emulsified in a Squaline/Arlacel vehicle containing nor-MDP(N-acetyl-glucosamine-3 yl-acetyl L-alanyl-D-isoglutamine). Subsequentbooster injections were given at 3 and 6 weeks after primaryimmunization. Rabbit sera was obtained weekly and purified, andquantified by ELISA (FIG. 7).

Following the purification of anti-VEGF and anti-EG-VEGF antibodies, wewent on to determine whether the elicited antibodies had anti-angiogenicproperties. Previous data demonstrates that ovarian function is tightlyregulated through angiogenic stimuli, and VEGF has been shown to beimportant in the recruitment and selection of follicles. We thushypothesized that if the antibodies were functioning as anti-angiogenicmolecules, then inhibition of follicle selection and growth and estrouscycle disruption would be expected with neutralization of VEGF.

Thirteen female C57BL/6 mice were injected intraperitoneally with 25 μgof purified anti-VEGF antibody every 3 days for 15 days. Estrous cycleswere monitored by obtaining daily vaginal smears, and the estrous cyclesof treated mice were compared with control mice injected withnonspecific IgG antibodies. In keeping with the anti-angiogenic effectof elicited anti-VEGF antibodies, a significant disruption of theestablished estrous cycle was seen beginning day 3 followingimmunization (FIG. 8A). The average length of the estrous cycles wasalso significantly decreased by over 66% (FIG. 8B). Primordial folliclegrowth was assessed at day 3 and day 7 following immunization byharvesting the ovaries of euthanized mice. A significant and persistentreduction in the number of primordial follicles (p<0.05 for bothcomparisons) was demonstrated in mice treated with anti-VEGF antibodies,suggesting potent anti-angiogenic efficacy of the VEGF antibodies (FIG.8C).

Collectively, these data demonstrated that immunogenic epitopes of VEGFand EG-VEGF can be identified and synthesized, and immunization leads toproduction of anti-VEGF and anti-EG-VEGF antibodies. Such antibodies arebiologically active, and function as anti-angiogenic molecules.

Determination of Immunogenicity of Anti-VEGF and Anti-EG-VEGF AntibodiesElicited from Active Immunization with Peptide Vaccines

ELISA demonstrated anti-VEGF and anti-EG-VEGF peptide antibodies torecognize the respective recombinant human VEGF (FIG. 9A) or EG-VEGFprotein (FIG. 9B). In this experiment, recombinant proteins were used tocoat ELISA plates, and the appropriate anti-peptide antibody was used.

Furthermore, we demonstrated that the anti-VEGF and anti-EG-VEGF peptideantibodies recognize rhVEGF by Western blot, with the resultant bands inthe expected location (42 kD, dimer) for VEGF blotted with anti-VEGFpeptide antibody (FIG. 10A) and VEGF monoclonal antibody (NeoMarkersAb-4, FIG. 10B). Likewise, a Western blot with rhEG-VEGF blotted withanti-EG-VEGF peptide antibodies (FIG. 10C) and a combinationanti-VEGF/anti-EG-VEGF peptide antibody (FIG. 10D) resulted in a band inthe expected location (22 kD, dimer) for rhEG-VEGF.

Assessment of in Vitro Biologic Effect of Anti-VEGF and Anti-EG-VEGFPeptide Antibodies

Following demonstration of the antigenic and immunogenic properties ofour anti-peptide antibodies, we went on to evaluate the functionalproperties of these molecules. In an effort to determine the effect ofour anti-VEGF peptide antibodies on the interaction of VEGF with theVEGF receptor, we used the Fluorokine assay. Briefly, washed cells areincubated with the biotinylated cytokine that in turn binds to the cellsvia specific cell surface receptors. The cells are then directlyincubated with avidin-fluorescein, which attaches to the receptor boundbiotinylated cytokine. Unbound biotinylated cytokine participates in anamplification reaction with the bound cytokine that results in anenhanced signal without compromising specificity.

Cells expressing the specific cytokine receptors are fluorescentlystained, with the intensity of staining proportional to the density ofthe receptors. Relative receptor density is then determined by flowcytometric analysis using 488 nm wavelength laser excitation. Throughthis experiment, we initially standardized the flow cytometry componentof the assay (FIG. 11A), in which different cell populations wereidentified using the kit supplied positive and negative controls.Following standardization, we used HUVEC cells that proliferate underthe influence of VEGF to determine the efficacy of binding of ouranti-peptide antibodies to VEGF, resulting in disruption of the normalVEGF-VEGF receptor interaction. As can be seen in FIG. 11B, a shift inpopulation toward decreased receptor density is demonstrated whenanti-VEGF peptide antibodies are used, suggesting a disruption in thenormal VEGF-VEGF receptor interaction.

Assessment of Anti-Angiogenic in Vivo Biologic Effect of Anti-VEGFPeptide Vaccines

Although we have demonstrated the interaction between VEGF and ouranti-peptide VEGF antibodies, it is still important to determine whetherthe effect of this interaction leads to a modification of angiogenicproperties of VEGF. To determine the anti-angiogenic properties of ourantibodies, we injected subcutaneous matrigel (basement membranegenerated from EHS sarcoma) into immunocompetent C57/BL6 mice, with orwithout rhVEGF. The matrigel was removed after 7 days, and cryostatsections were cut and stained with a conjugate of CD31 (which Binds toendothelial cells) and phycoerythrin. Angiogenesis was qualitatively andquantitatively determined through fluorescence confocal microscopy. Theaddition of rhVEGF to the matrigel led to a significant increase inangiogenesis relative to control.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification, all of whichare hereby incorporated by reference in their entirety. The embodimentswithin the specification provide an illustration of embodiments of theinvention and should not be construed to limit the scope of theinvention. The skilled artisan recognizes that many other embodimentsare encompassed by the claimed invention and that it is intended thatthe specification and examples be considered as exemplary only, with atrue scope and spirit of the invention being indicated by the followingclaims.

What is claimed is:
 1. A peptide comprising: a) at least one VEGFepitope selected from the group consisting of amino acids 4 through 21of SEQ ID NO: 1, amino acids 24 through 38 of SEQ ID NO: 1, amino acids76 through 96 of SEQ ID NO: 1, amino acids 126 through 143 of SEQ ID NO:1, amino acids 127 through 144 of SEQ ID NO: 1, amino acids 162 through175 of SEQ ID NO: 1, amino acids 5 through 15 of SEQ ID NO: 2, aminoacids 24 through 34 of SEQ ID NO: 2, amino acids 50 through 75 of SEQ IDNO: 2, amino acids 50-67 of SEQ ID NO: 2, and amino acids 86 through 105of SEQ ID NO: 2; b) a helper T cell epitope; and c) a linker joining theat least one VEGF epitope to the helper T cell epitope, wherein thelinker comprises from about 2 to about 10 amino acids.
 2. The peptideaccording to claim 1, having from about 35 to about 70 amino acids. 3.The peptide according to claim 1, wherein the helper T cell epitope is apromiscuous Th cell epitope.
 4. The peptide according to claim 1,wherein the helper T cell epitope comprises from about 14 to about 22amino acids.
 5. The peptide according to claim 1, wherein the helper Tcell epitope comprises a sequence chosen from TT (SEQ ID NO: 3), TT1(SEQ ID NO: 4), P2 (SEQ ID NO: 5), P30 (SEQ ID NO: 6), MVF (SEQ ID NO:7), HBV (SEQ ID NO: 8), and CSP (SEQ ID NO: 9).
 6. The peptide accordingto claim 1, wherein the linker comprises Gly-Pro-Ser-Leu (SEQ ID NO:10).
 7. An immunogenic composition comprising at least one immunogenchosen from at least one of the peptides according to claim 1, and atleast one pharmacologically acceptable carrier.
 8. A polynucleotidewhich encodes one of the peptides according to claim 1.